Bioscience, Biotechnology, and Biochemistry

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Salkowski’s Reagent Test as a Primary Screening

Index for Functionalities of Rhizobacteria Isolated
from Wild Dipterocarp Saplings Growing Naturally
on Medium-Strongly Acidic Tropical Peat Soil

Atiqur RAHMAN, Irnayuli R. SITEPU, Sui-Yan TANG & Yasuyuki HASHIDOKO

To cite this article: Atiqur RAHMAN, Irnayuli R. SITEPU, Sui-Yan TANG & Yasuyuki HASHIDOKO
(2010) Salkowski’s Reagent Test as a Primary Screening Index for Functionalities of Rhizobacteria
Isolated from Wild Dipterocarp Saplings Growing Naturally on Medium-Strongly Acidic Tropical
Peat Soil, Bioscience, Biotechnology, and Biochemistry, 74:11, 2202-2208, DOI: 10.1271/
bbb.100360

To link to this article: https://doi.org/10.1271/bbb.100360

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Biosci. Biotechnol. Biochem., 74 (11), 2202–2208, 2010

Salkowski’s Reagent Test as a Primary Screening Index for Functionalities

of Rhizobacteria Isolated from Wild Dipterocarp Saplings Growing Naturally
on Medium-Strongly Acidic Tropical Peat Soil
Atiqur R AHMAN,1 Irnayuli R. S ITEPU,2 Sui-Yan T ANG,1 and Yasuyuki H ASHIDOKO1; y
1
Division of Applied Bioscience, Research Faculty of Agriculture, Hokkaido University,
Kita 9 Nishi 9, Kita-ku, Sapporo 060-8589, Japan
2
Forest Microbiology Laboratory, Forest and Nature Conservation Research and Development Center,
Forest Research and Development Agency, Jalan Gunung Batu No. 5, P.O. Box 165, Bogor 16610, Indonesia

Received May 12, 2010; Accepted June 28, 2010; Online Publication, November 7, 2010
[doi:10.1271/bbb.100360]

Rhizobacteria isolated from wild dipterocarp saplings
in Central Kalimantan, Indonesia, were subjected to
Salkowski’s reagent test, which is often used in detect-
ing indolic substances. Among 69 isolates grown in a
low-nitrogen medium supplemented with L-tryptophan
(TRP), culture fluids of 29 strains were positive to the
test, in which 17 bacteria turned red and other 10 pink.
All the red type rhizobacteria actively converted TRP
into tryptophol (TOL), while some yielded indole-3-
acetic acid (IAA) with TOL production. They also
showed a capacity to decompose gallotannin into pyro-
gallol via gallic acid. On the other hand, an active IAA-
producing Serratia sp. CK67, and three Fe-solubilizing
Burkholderia spp. CK28, CK43, and Citrobacter sp. CK42,
were all involved in pink type rhizobacteria, which were
more effective, oxidative TRP-degraders than the red
type rhizobacteria. Thus, Salkowski’s reagent test
should be a useful primary index in the screening of
functional rhizobacteria in peatland ecosystem.
Key words: Salkowski’s reagent test; functional rhizo-
bacteria; tropical peat soil; dipterocarp; IAA
production
Tropical swamp forest yields woody peat soil in the
forest bed, and this medium-strongly acidic soil is rich in
polyphenols, enough to prevent a good growth of tree
seedlings. During past decade and present, peat swamp
forests in Central Kalimantan had been and are still
disappearing rapidly due to excessive deforestation and
peat fire. Although many attempts at reforestation to
rescue natural forest vegetation have been made, many
trials, including transplanting of nursery plants of local
trees, were not successful due to cost problem, the
properties of the soil, and unprofitable land manage-
ment.1) Hence it is necessary to find a new approach to
the rehabilitation of destroyed peatland. One practical
trial using biological tools has been done by our group to
search in the rhizosphere for functional microorganisms
that assist the growth of tree seedlings and saplings.2,3)
Using naturally grown seedlings and saplings of local
soil-adapted dipterocarp trees, including some local
Shorea spp., Dipterocarpus spp., and Hopea spp., as
sources of functional rhizospherous microbes, we have
investigated the functional rhizobacteria known as PGPR
(plant growth-promoting rhizobacteria) for dipterocarp
seedling.
A preliminary study indicated that these growth-
promoting bacteria stimulated the development of root
systems. In fact, many groups have reported an associa-
tion of indole-3-acetic acid (IAA) with PGPR.4–6) It is
known that appropriate concentrations of exogenous
IAA stimulate the growth and development of plant root
systems.7) Hence we speculated that some active PGPRs
in peatland provide IAA or IAA agonists, which activate
root branching and lateral root development. Among the
rhizobacteria, active isolates showed positive reactions
to Salkowski’s reagent test, with a pinkish or a deep
red coloration.8) Salkowski’s reagent is a 35% HClO4
solution containing 10 mM FeCl3 , and when mixed with
IAA, tris-(indole-3-acetato)iron(III) complex is formed
to display pink coloration.9) These positive reactions of
test bacteria indicate their capability of metabolizing
L-tryptophan (TRP) to IAA or some analogous com-
pounds of IAA. As exogenous IAA in the rhizosphere is
a key phytohormone-like chemical substance for root-
microbe interaction, the bio-conversion process of TRP
to IAA in bacteria has been investigated well following
Perley and Stowe.10) Particularly, TRP transaminase,
which catalyzes an oxidative deamination on TRP to
yield indole-3-pyruvic acid (IPyA), was first reported in
Enterobacter cloacae, which showed a remarkable
capability of IAA production.11,12) Hence Salkowski’s
reagent test, specified for the detection of IAA and its
precursory compound IPyA, is often used for primary
screening of IAA-producing rhizobacteria.
Another important factor in plant growth on peatland
is trace elements, particularly Fe and Cu.13,14) The
deficiency of such metal cations in tropical peat soil is
due to the polyphenolic acid-rich peat soil that possesses
catechol and carboxy groups to capture these metallic
cations.15) Rhizobacteria having polyphenol-degrading
abilities can decompose and quench phenolic substances
present in the rhizosphere of local dipterocarp seedlings,
leading to activation of their growth on tropical peat
soil. In addition, rhizobacteria that can produce metal
cation-chelating agents probably assist in the uptake of
trace elements by local tree seedlings. Thus, peat soil-

y
To whom correspondence should be addressed. Tel: +81-11-706-3840; Fax: +81-11-706-4182; E-mail: yasu-h@abs.agr.hokudai.ac.jp
 Salkowski’s Reagent in Rhizobacterial Search
COOH
R subjected to extraction of chromosomal DNA by the use of an Isoplant
NH2
N the 16S rRNA gene region, sequencing of the PCR product by a direct
H N
H
R = COOH, indole-3-acetic acid (IAA)
R = CH2OH, tryptophol (TOL) L-tryptophan (TRP)
R = CH2OCOCH3, O-acetyltryptophol
R = CH(OH)COOH, indole-3-lactic acid (ILA)
R = COCOOH, indole-3-pyruvic acid (IPyA)
Fig. 1. Chemical Structures of Indolic Compounds.
adapting local dipterocarp saplings are a good source of
multifunctional rhizobacteria, and therefore, simple but
effective screening for such multifunctional rhizobac-
teria is awaited. In this study, colorimetric responses to
Salkowski’s reagent test, which also shows oxidative
degradation of the indolic amino acid, were investigated
as a possible primary index of polyphenol-degrading and
Fe-chelating rhizobacteria, along with IAA-production.
Materials and Methods
General. TLC plates (Merck Kieselgel 60F254 Art-5715, 0.25 mm
thick, Merck, Whitehouse Station, NJ) were used for preparative and
analytical TLC. L-Tryptophan (TRP), indole-3-acetic acid (IAA),
indole-3-lactic acid (ILA), indole-3-ethanol (TOL), and indole-3-
carboxylic acid, purchased from Wako Pure Chemical Industries
(Osaka, Japan), were used as authentic compounds. FeCl3 and HClO4
to prepare Salkowski’s reagent were also from Wako. 1 H and
13
C NMR analyses were recorded on a Bruker AMX-500 (Bruker,
Rheinstetten, Germany) spectrometer working at 500 and 125 MHz
respectively. To measure NMR spectra, acetone-d6 , CDCl3 , CD3 OD,
and DMSO-d6 (Sigma-Aldrich, St. Louis, MO and Wako) were used.
Mass spectra were recorded on a JEOL JMS-SX102A (JEOL, Tokyo)
mass spectrometer for FAB-MS and FD-MS.
Culture medium and growth conditions. The media for trapping
microbes and separation of single colony were described in our previous
paper.1) The basal medium for TRP metabolism was Winogradsky’s
mineral solution containing 1.5% sucrose and 0.005% yeast extract to
which TRP (100 mg l
1 ) was added. It was adjusted to pH 6.0–6.2. As
the inoculant, three loops of test bacterium precultured on a modified
Winogradsky’s agar (MWA) plate16) for 3 d at 20  C were suspended in
2 ml of sterile water (OD660 0.6). For the preliminary coloration assay,
10 ml-medium was poured into a 50-ml Erlenmeyer flask, while
200 ml-medium was used in a 300-ml Erlenmeyer flask for extraction
and characterization of IAA-metabolites. To the medium in a 300-ml
Erlenmeyer flask, 100 ml of bacterial cell suspension was inoculated,
and this was standing-cultured at 28  C in the dark for 7 d. For aerobic
culture, 200 ml of TRP-containing medium was shaking-cultured in a
500-ml shaking flask at 100 rpm (Bio-Shaker BR-300LF, Taitec,
Nagoya, Japan) at 28  C in the dark for 7 d. For active aromatic
compound-degraders, a shortened culturing period (2 d) was also
tested.
Bacterial isolates. Bacterial isolates were obtained from the
rhizosphere of dipterocarp seedlings and saplings collected in Central
Kalimantan, Indonesia.1) The roots were then inoculated into nitrogen-
deficient soft gel medium of Winogradsky’s mineral solution base and
pre-cultured at 25  C in the dark for 3 d. The trapping-cultured medium
was spread over an MWA plate, and single colonies were purified as
described in a previous paper.17) Isolates grown on MWA were stored
at
80  C as 10% glycerol cultures.
Sequence determination of the bacterial 16S rRNA gene. In some
cases, a trace amount of bacterial colony was used directly as a
template for PCR for the 16S rRNA gene. When this was unsuccessful,
rhizobacterium grown overnight in nutrient broth medium was
2203
collected by centrifugation, and the resulting bacterial cell pellet was
II DNA extraction kit (Nippon Gene, Toyama, Japan). Amplification of
PCR method, and searching in a database program were as described
previously.17)
Salkowski’s reagent test for colorimetric IAA detection. Some
selected bacterial isolates were subjected to qualitative screening for
IAA-producing bacteria using Salkowski’s reagent (2% of 0.5 M FeCl3
in 35% HClO4 solution) for colorimetric IAA detection in liquid
culture.8) A bacterial culture incubated in 200 ml Winogradsky’s
medium supplemented with 100 mg l
1 TRP for 7 d without any
shaking was centrifuged at 10,000 rpm for 10 min at 4  C, and 1 ml of
the supernatant was mixed with 2 ml of Salkowski’s reagent in a test
tube. This was kept at room temperature in the dark for 30 min. IAA
production in the cultured medium was evident by a characteristic
indication of reddish to pinkish color in the solution, while TRP
showed a pale yellow or colorless response. According to the color
intensity of the test solutions, coloring responses were grouped into
two: pink type (pale pink to deep pink) and red type (dark red to red).
One ml of an aqueous solution of 0.25 mM IAA, 0.25 mM TOL, or
0.25 mM IAA plus 0.25 mM TOL in water mixed with 2 ml Salkowski’s
reagent was measured directly for UV spectrum (U3310 Spectropho-
tometer, Hitachi, Tokyo) in a range from 220 to 650 nm (Supplemental
Fig. S1, see Biosci. Biochem. Biotechnol. Web site).
Extraction, detection, and quantification of indole derivatives on
TLC and HPLC. Salkowski’s reagent was also used as a chromogenic
spraying reagent to detect IAA and other indole derivatives derived
from TRP on TLC plates.18) IAA, TOL, ILA, and TRP were used as
authentic indole derivatives, while IPyA was confirmed by 1 H NMR
spectra of the EtOAc extract from a 2-d shaking culture of CK67. After
7 d of incubation in standing culture, cultured media were centrifuged
at 10,000 rpm for 10 min, and the resulting supernatant was acidified
with 2 M HCl to pH 2.5.2) The supernatants were extracted with equal
volumes of EtOAc three times. After it was concentrated and re-
dissolved in 2.0 ml EtOAc, the volumetric solution was used in semi-
quantitative analysis of the TRP metabolites. As a preliminary, 5 ml
was exactly charged on TLC using a volumetric glass capillary. Indole
compounds were detected on silica gel TLC plates developed in
CHCl3 -EtOAc-HCOOH at 11:5:4. Chemical spots observed under UV
254 nm were sprayed with Salkowski’s reagent and then slightly
heated. Each indole compound showed characteristic, stable colora-
tion: reddish pink for IAA, reddish yellow for TRP, and vivid yellow
for TOL and ILA.
HPLC was done to quantify the major TRP metabolites, IAA, TOL,
ILA, and tentative IPyA, in a reverse-phase column (L-column2 ODS
5 mm, 4.6 mm i.d.  250 mm, Chemicals Evaluation and Research
Institute, Tokyo) using MeOH: 1% CH3 COOH, 25:7519) as an isocratic
mobile phase, with a UV detector at 280 nm. The retention times of
TRP, ILA, TOL, IPyA, and IAA were tR 6.3, 22.8, 29.0, 30.9, and
33.5 min respectively (Figs. 1 and 2). The absolute calibration method
was used for the HPLC quantification of IAA to give a coefficient of
0:6  103 (as peak intensity per ng IAA).
Purification of indole compounds from the crude extracts was done
by silica gel column chromatography, and continuously by preparative
thin layer chromatography using silica gel TLC plates. All the major
metabolites (IAA, TOL, ILA, and catechol), except for IPyA, were
purified by column chromatography, and each structure was confirmed
by 1 H and mass spectroscopic (FD-HR-MS or FAB-HR-MS) analysis.
TRP metabolism in different pH. Bacterial isolates CK23 and CK67
were tested to determine whether these bacteria are equally capable of
metabolizing TRP at various pH levels. An MW solution supplemented
with 100 mg l
1 TRP was adjusted to pH 4.0, 5.0, 6.0, 7.0, and 8.0 using
2 M H2 SO4 . To 100 ml of the TRP-amended medium, a loop of bacteria
was inoculated and standing-cultured for 7 d in the dark at 28  C. The
process of extracting TRP metabolites was as described above.
Tannic acid degradation. Since both the tropical woody peat soil
and the root bark of dipterocarp plant were rich in diverse
polyphenols,20,21) we also investigated the tannic acid (TA)-degrading
2204 A. R AHMAN et al.
Table 1. Isolation of Rhizobacteria from Dipterocarp Saplings

Isolation source Code Identification Subclass Response

(Pink type)
Hopea sp. CK2 Pseudomonas sp.
-Proteobacteria pale pink
CK42 Citrobacter sp.
-Proteobacteria pink
Shorea parvflora CK13 Enterobacter sp.
-Proteobacteria pale pink
S. teysmanniana CK26 Azospirillum sp. -Proteobacteria pink
CK28 Burkholderia sp. -Proteobacteria pale pink
CK59 Burkholderia sp. -Proteobacteria pale pink
CK61 Citrobacter sp.
-Proteobacteria pink
S. balangeran CK43 Burkholderia sp. -Proteobacteria pale pink
Dipterocarpus sp. CK22 Serratia sp.
-Proteobacteria pale pink
CK67 Serratia sp.
-Proteobacteria pale pink
(Red type)
Hopea sp. CK15 Roseateles sp.
-Proteobacteria red
CK40 Klebsiella sp.
-Proteobacteria deep red
Dipterocarpus sp. CK23 Enterobacter sp.
-Proteobacteria red
CK24 Erwinia sp.
-Proteobacteria deep red
CK34 Stenotrophomonas sp.
-Proteobacteria red
CK35 Enterobacter sp.
-Proteobacteria deep red
CK36 Pantoea sp.
-Proteobacteria deep red
Shorea balangeran CK4 Klebsiella sp.
-Proteobacteria deep red
CK5 Serratia sp.
-Proteobacteria deep red
CK6 Klebsiella sp.
-Proteobacteria deep red
CK64 Enterobacter sp.
-Proteobacteria deep red
CK65 Enterobacter sp.
-Proteobacteria deep red
S. parvflora CK10 Erwinia sp.
-Proteobacteria deep red
CK12 Erwinia sp.
-Proteobacteria deep red
S. teysmanniana CK53 Pantoea sp.
-Proteobacteria deep red
CK54 Pantoea sp.
-Proteobacteria deep red
S. stenoptera CK69 Enterobacter sp.
-Proteobacteria deep red
(Intermediary type)
Shorea teysmanniana CK18 Klebsiella sp.
-Proteobacteria pale red
CK19 Rhizobium sp. -Proteobacteria pale red
(Colorless, two selected isolates)
Shorea balangeran CK32 Burkholderia sp. -Proteobacteria colorless
S. teysmanniana CK63 Pseudomonas sp.
-Proteobacteria colorless
Salkowski’s reagent test was done at 10-ml volume in a 50-ml Erlenmeyer flask.

capability of the rhizobacteria. To 100 ml of modified Winogradsky’s
liquid medium supplemented with TRP, TA (averaged molecular
weight, 1,700, 1.7 g l
1 ) was added as model polyphenolic to
approximately 1 mM concentration, and all 29 rhizobacteria were
shaking-cultured for 2 d as described above. The supernatant of the
cultured medium was adjusted to pH 3.0 or less with 1 M HCl, and
extracted twice with equal volumes of EtOAc. The culture fluid with or
without 1 mM TA was extracted with EtOAc as described in this paper,
and EtOAc solubles were analyzed by the TLC system, with spraying
of Salkowski’s reagent or Gibbs reagent to detect polyphenols.22)
Isolation and identification of the major bio-conversion products from
TA were done as described in a previous paper.23)
Detection of IAA in the culture fluid of IAA-producing Serratia sp.
CK67 standing-cultured in liquid medium without supplemental TRP.
Serratia sp. CK67, an active IAA-producing bacterium, was used in
this experiment. The medium (200 ml), supplemented with 50 mg l
1
yeast extract and 0.05% NH4 NO3 (0.5 g l
1 ), was standing-cultured for
3 weeks, and after separation of bacterial cells by centrifugation at
8;000  g for 10 min, the culture fluid was passed through conditioned
Cosmosil 75C18-OPN (120 g, Nacalai Tesque, Kyoto, Japan) on a
Buchner funnel, and this was then washed with a sufficient volume of
Milli-Q water (200 ml  3). After removal of the void water by
vacuum, the substances trapped by the Cosmosil particles were eluted
with 600 ml MeOH. Then the resulting eluate was concentrated and
redissolved to 2 ml-volumetric solution, of which 5 ml was subjected to
semi-quantitative TLC and quantitative HPLC analyses.
Plate assay for bacterial siderophores using chrome azurol S
(CAS). Screening of bacteria able to produce Fe-chelating substances
was done using chrome azurol C (CAS)-containing agar plates, as
described in the original paper.24) A basal medium, 100 ml-MM9 salt
solution (3 g l
1 KH2 PO4 , 5 g l
1 NaCl, and 10 g l
1 NH4 Cl; 10),
30.24 g of PIPES, and 12 ml of 50% NaOH solution mixed in 750 ml
of Milli-Q water, was solidified with 1.5% agar and autoclaved. Into
the liquefied medium, a CAS-Fe(III)-containing hexadecyltrimethyl
ammonium bromide solution was gently mixed and casted on Petri
dishes. Since the basal MM9 medium contained sufficient amounts of
phosphate and ammonium, some oligotrophic bacteria grew well on
the plate. Hence each bacterial isolate was inoculated on the CAS plate
by means of toothpicks. A positive response in the CAS assay gave a
clear, yellowish halo-zone around the grown colony on a blue
background.
Results and Discussion
In our investigation of functional rhizobacteria from
collected saplings of Shorea spp., Hopea sp., and
Dipterocarpus sp. growing naturally in tropical peatland
in Central Kalimantan, Indonesia, many isolates showed
PGPR activity for S. balangelan and S. selanica seed-
lings.3) Of the 69 bacterial isolates from the rhizosphere
of these dipterocarp saplings, approximately half (34
isolates) showed positive color reactions to Salkowski’s
reagent. According to coloration, these positive isolates
were grouped into three types: a pink type (CK2, 13, 22,
26, 28, 42, 43, 59, 61, and 67), a red type (CK4, 5, 6, 10,
12, 15, 23, 24, 34, 35, 36, 40, 53, 54, 64, 65, and 69), and
 Salkowski’s Reagent in Rhizobacterial Search 2205

Standard Table 2. Bio-Conversion of TRP by Mainly Two Groups of

CK67 standing-culture

Concentration as IAA (µg/ml medium)

Concentration as IAA (µg/10 µl std. sln.)

Rhizobacteria to Show Different Responses toward Salkowski’s

(7 d)
TRP
Reagent Test

IAA
10

TOL
IAA
0.2
Standing-culture Shaking-culture
Isolate
ILA IAA TOL IAA IPyA
5 (Pink type)

IPyA
0.1
Pseudomonas sp. CK2

ILA
Enterobacter sp. CK13



Serratia sp. CK22

þþ 
Azospirillum sp. CK26 



0 10 20 30 40 0 10 20 30 40 Burkholderia sp. CK28




Time (min) Time (min)
Citrobacter sp. CK42
 þþ 
Burkholderia sp. CK43




CK67 shaking-culture CK67 shaking-culture Burkholderia sp. CK59

þ

Concentration as IAA (µg/ml medium)

Concentration as IAA (µg/ml medium)

(7 d) 6 (2 d) Citrobacter sp. CK61

þþ 

IPyA
IAA

10 Serratia sp. CK67 þþ
þþ þ

IAA
4 (Red type)
Klebsiella sp. CK4 þþ þþ þ þ
IPyA

5 Serratia sp. CK5 þþ þþ þþ þ

2 Klebsiella sp. CK6 þþ þþ þ þ
Erwinia sp. CK10  þþ þ 
Erwinia sp. CK12  þþ þ 
Roseateles sp. CK15
þþ þ þþ
0 10 20 30 40 0 10 20 30 40 Enterobacter sp. CK23  þþ þ þþ
Time (min) Time (min) Erwinia sp. CK24
þþ þ 
Stenotrophomonas sp. CK34




Fig. 2. HPLC Profiles of TRP Metabolites Yielded by IAA-Produc- Enterobacter sp. CK35

NT
ing Serratia sp. CK67 under Aerobic and Anaerobic Conditions. Pantoea sp. CK36
þþ NT
For the anaerobic condition, standing culture was selected, while Klebsiella sp. CK40

NT
shaking culture was set at 100 rpm for the aerobic conditions, and Pantoea sp. CK53
þþ
þ
liquid media of 200 ml were cultured for 7 d. Milli-Q water Pantoea sp. CK54 
þ þþ
containing 0.25 mM TRP and 0.5 mM ILA, TOL, and IAA was used Enterobacter sp. CK64

þþ þþ
as standard solution, of which 10 ml was subjected to HPLC.
Enterobacter sp. CK65

NT
Identification of the major peaks in these HPLC profiles at 280 nm
Enterobacter sp. CK69

NT
was done by a double injection of standard IAA to detect major
substance at the same retension time. Compared to the 7-d standing
culture and the 7-d-shaking culture, 2-d-shaking culture maintained (Intermediary type)
IPyA. All the extracts were re-dissolved in 2.0 ml MeOH, and 5 ml of Klebsiella sp. CK18 þþ þ þ þþ
the volumetric solution was subjected to HPLC analysis. The scale Rhizobium sp. CK19 þþ þ þ þþ
of the vertical axis shows the concentration of IAA in the cultured
medium (mg/ml) as a representative indolic compound. (Colorless type)
Burkholderia sp. CK32   

Pseudomonas sp. CK63




an intermediary type (CK18 and 19), and tentative þþ, major spot (over 5 mg/ml medium); þ, minor spot; , trace spot;
, not
identification at the genus level by determination of 16S detected; NT, not tested. Both in standing culture and shaking culture,
rRNA gene sequence (Table 1) was done. Two bacteria incubation was done for 7 d.
(CK32 and 63) that responded negatively to Salkowski’s
reagent were also selected for comparisons to the posi- aerobic-cultured for 7 d, gave IAA as the major meta-
tive ones. Generally, bacteria grown under nitrogen- bolite from TRP (Supplemental Fig. 2; see Biosci.
limiting conditions enhance the level of deaminase Biotechnol. Biochem. Web site). Including Serratia sp.
for effective recycling of nitrogen from amino acids,25) CK67, they had no or low ability to degrade IAA (data
but some bacteria use transaminase for the oxidative not shown), and thus it is plausible that they accumulated
deamination reaction on L-tryptophan (TRP) to yield IAA in the TRP-supplemented cultured medium. All
IAA via intermediary indole-3-pyruvic acid (IPyA).26) these effective IAA-producing bacteria are members of
Although TRP contains two nitrogen atoms, an N in the the pink type rhizobacteria (Table 2). None of the
indole ring is less degradable than the other N in the members of this group produced tryptophol (TOL) or
-amino group. Therefore, active saprophytes possess- DL-indole-3-lactic acid (ILA) under anaerobic culture
ing indole oxidase completely degrade TRP via anthra- conditions.
nilic acid and then catechol,27) and do not produce In contrast, the red type rhizobacteria, Klebsiella spp.
indole-3-acetic acid (IAA). CK4 and CK6 and Serratia sp. CK5, effectively
Among the rhizobacteria positive to Salkowski’s converted TRP into TOL with active IAA production,
reagent, Serratia sp. CK67 was selected as the most particularly in standing culture (CK4 and CK6 in
selective and effective IAA-producer from exogenous Fig. 3). Under the same anaerobic culture conditions,
TRP under both aerobic and anaerobic culture conditions Erwinia sp. CK10, CK12, and CK24, Enterobacter sp.
(Table 2 and Fig. 2). Compared to aerobic culturing for CK23, and Pantoea spp. CK36 and CK53 yielded TOL
7 d, 2-d-aerobic culturing resulted in a peak at tR and ILA, without production of IAA (see CK12 and
28.5 min in HPLC identical to IPyA. Similarly, Serratia CK23 selected as typical TOL-ILA producing rhizobac-
CK22, Citrobacter sp. CK42, and Citrobacter sp. CK61, teria) (Fig. 3). Thus TOL production under anaerobic
2206
Concentration as IAA (µg/ml medium)
30 CK4
(IAA-TOL)
20
10
0 10 20 30
Time (min)
Concentration as IAA (µg/ml medium)
CK12
30 (TOL)
20
10
0 10 20 30
Time (min)
Fig. 3. HPLC Profiles of Anaerobic TRP Metabolites Yielded by
TOL-ILA-Producing Rhizobacteria of the Red Type toward Sal-
kowski’s Reagent.
All the HPLC profiles were under anaerobic culture conditions for
7 d. For standard compounds as references, the profile of the
standard mixture shown in Fig. 3 should be used. All the extracts
from the culture fluid of the red type rhizobacteria were re-dissolved
in 2.0 ml MeOH. The volumetric solution was diluted 10 times with
fresh MeOH, of which 5 ml was subjected to HPLC analysis. (IAA-
TOL) in the figure means a metabolic pattern predominantly
producing both IAA and TOL, while (TOL-ILA) shows a pattern
producing TOL and ILA but not IAA. (TOL) produces only TOL.
culture conditions appears to be characteristic of the red
type rhizobacteria. Even in shaking culture for 7 d, some
isolates produced TOL as a major product, together with
significant amounts of IAA (see CK5 and CK19 selected
as typical TOL-IAA producing rhizobacteria under
aerobic conditions) (Supplemental Fig. 3; see Biosci.
Biotechnol. Biochem. Web site).
TOL is known to be derived from endogenous IAA by
plants, and it is immediately converted to O-acetyl
tryptophol, which is stocked in plant tissues.28) TOL is
also known as an antimicrobial substance and a weak
auxin agonist,29) and it has been identified as an
apoptosis-inducing factor in a human leukemic cell
line.30) Hence, it might be necessary to investigate the
association of TOL with plant apoptosis.
In bio-conversion experiments at various pH levels
(pH 4.0, 5.0, 6.0, 7.0, and 8.0 before autoclaving) for
five isolates selected (pink type, Azospirillum sp. CK26
and Serratia sp. CK67; red type, Klebsiella sp. CK6 and
Enterobacter sp. CK23; and a colorless type, Burkhol-
deria sp. CK32), all the rhizobacteria except for Serratia
sp. CK67 showed good growth performance at all pH
values. Klebsiella sp. CK6 uniquely produced O-acetyl
tryptophol at pH 5.0 as a major metabolite from TRP,
although TOL was major substance produced at pH 6.0
and 7.0 (Fig. 3). In contrast, Serratia sp. CK67,
selectively producing IAA, did not grow at pH 5.0 or
lower, but grew well in a range from pH 6.0 to 8.0. The
A. R AHMAN et al.
IAA production of CK67 was uniquely active at pH 7.0
Concentration as IAA (µg/ml medium)
CK6 but inactive at pH 8.0. Peat soil-adapted local trees
(IAA-TOL) maintain their rhizosphere pH in the neutral region
40
(pH 6.0–7.2), although tropical peat soil itself indicates
more acidic, ranging from pH 3.8–4.5.31) The appropri-
ate pH range for the IAA production of CK67 suggests
20 that Serratia sp. CK67 is a genuine rhizobacterium
preferably inhabiting the rhizosphere of dipterocarp
saplings to assist the growth of the host plants.
As polyphenols and humic acid cut down on the
40 0 10 20 30 40
availability of phosphorus and trace elements, tropical
Time (min) peat soil that is extremely rich in these aromatic
substances often inhibits plant growth.13,14) If rhizos-
Concentration as IAA (µg/ml medium)
CK23 pherous bacteria can degrade such pholyphenols and
30 (TOL-ILA) humic substances, they can be important partners for
local plants inhabiting the peatland ecosystem. To
20 determine whether these polyphenol-degrading bacteria
contribute to reducing the adversity of tropical peat soil
in the rhizosphere, a polyphenol-metabolizing experi-
10
ment was done under a shaking-culture condition with
exposure to 1 mM tannic acid (TA). As the culture
medium, the medium used for TRP metabolizing assay
40 0 10 20 30 40 was subjected to this experiment because TRP is an
Time (min) available nitrogen source. Among 15 red type and 2
intermediary type rhizobacteria isolated from polyphe-
nol-rich environments, 11 isolates produced pyrogallol
(PY) from TA via non-oxidative decarboxylation of
precursory gallic acid (GA) (Table 3). On the other
hand, the responses of the pink type rhizobacteria to
TA-degradation were clearly distinguishable from those
of the red ones. After 2 d of incubation under aerobic
conditions, four pink type bacteria, Pseudomonas sp.
CK2, Enterobacter sp. CK13, Azospirillum sp. CK26,
and Burkholderia sp. CK43, thoroughly degraded TRP
to catechol (CA) via anthranilic acid.27) However, these
four isolates gave on TLC neither PY nor other
metabolites of TA. Furthermore, four other isolates
yielded GA as breakdown products of TA, but none of
them gave PY (Table 3). Thus, the color type of the
rhizobacteria in Salkowsky’s reagent test reflected their
metabolic properties for TA-degradation.
To confirm the practical contribution of IAA-produc-
ing rhizobacteria to dipterocarp seedlings in the peatland
ecosystem, it is also necessary to characterize stable
providers of TRP. In some studies, it has been reported
that significant amounts of TRP are exuded from the
roots of some plant seedlings, such as oat (21–29 mg/g
of 3-d-grown fresh seedlings)32) and radish (0.3–0.4
mg/seedling).33) In contrast, low exudation of TRP has
also been reported for some other plants, such as
cucumber, showing only 0.002 mg/seedling.34) Thus, the
capability of plant roots to exude TRP into the rhizo-
sphere is dependent on the plant species, along with
environmental conditions of the seedlings. Huang and
Villanueva have analyzed the amino acid contents of the
seedlings of some dipterocarp plants, but offered no
particular description for TRP.35)
Another possible source of TRP is the rhizobacteria
themselves. It is known that wild Corynebacterium
glutamicum and Bacillus subtilis effectively produce
TRP in culture,36) but many reports have also indicated
that TRP is the most costly biosynthesized amino acid
among 20 constitutive amino acids as protein compo-
nents.37) When we cultured IAA-producing Serratia sp.
 Salkowski’s Reagent in Rhizobacterial Search 2207
Table 3. Some Functionalities of Mainly Two Groups of Rhizobac- Standard CK67
teria According to Salkowski’s Reagent Test (without TRP)

Concentration as IAA (µg/ml medium)

Concentration as IAA (ng/µl std. sln)
Isolates GA PY CA CAS 50
0.1
(Pink type) 40
Pseudomonas sp. CK2

þþ 
Enterobacter sp. CK13

þþ  30
Serratia sp. CK22 þþ 

20
0.05
Azospirillum sp. CK26

þþ

Burkholderia sp. CK28
þþ  þþ 10
Citrobacter sp. CK42 þþ 
þþ 0
0
Burkholderia sp. CK43
 þþ þþ
Burkholderia sp. CK59


0 10 20 30 40 0 10 20 30 40
Citrobacter sp. CK61 þþ 

Time (min) Time (min)
Serratia sp. CK67 þ 


Fig. 4. HPLC Profiles of Metabolites Yielded by IAA-Producing
(Red type) Serratia sp. CK67 in Modified Winogradsky’s Culture Medium.
Klebsiella sp. CK4 þ þþ
 Solutions containing 2.5 mM TRP and 5 mM of ILA, TOL, and IAA
were used as standard solution, of which 5 ml was subjected to
Serratia sp. CK5 þþ þ


HPLC. Metabolites of standing-cultured CK67 for 3 weeks re-
Klebsiella sp. CK6 þ þþ


dissolved in 2.0 ml MeOH, of which 10 ml solution was subjected to
Erwinia sp. CK10 þ þþ

HPLC analysis. The scale of the vertical axis shows the concen-
Erwinia sp. CK12 þ þþ
 tration of IAA in the cultured medium (mg/ml) as the common
Roseateles sp. CK15 þ þþ
 indolic compound.
Enterobacter sp. CK23 þ þþ

Erwinia sp. CK24 þ þþ

Stenotrophomonas sp. CK34



Enterobacter sp. CK35 NT

Pantoea sp. CK36 NT


Klebsiella sp. CK40




Pantoea sp. CK53 þ þþ

Pantoea sp. CK54 þ þþ

Enterobacter sp. CK64 þþ 


Enterobacter sp. CK65 NT


Enterobacter sp. CK69 NT

(Intermediary)
Klebsiella sp. CK18 þþ þþ


Rhizobium sp. CK19 þþ þþ


(Colorless)
Burkholderia sp. CK32 þþ þ


Pseudomonas sp. CK63 þþ


GA, gallic acid; PY, pyrogallol; CA, catechol. Bio-conversion of TA into
GA and PY, and production of CA from TRP are shown here. GA
production suggests bacterial functionality to be tolerant to polyphenols and
to degrade hydrolysable tannins, while PY production shows non-oxidative
decarboxylation of gallic acid. CA reflects high anthrani activity to cleave
þþ, major; þ, minor; , trace;
, not detected. CAS indicates CAS assay
for bacterial siderophore. þþ, size of Fe-chelating halo-zone is over 5 mm;
þ, halo-zone is visible, but its size of less than 5 mm; , weak positive to
change the color only on edge of the colony;
, negative.
CK67 cultured in NH4 NO3 -amended Winogradsky’s
medium (0.005% yeast extract and 0.05% NH4 NO3 for
nitrogen source, 0.5% sucrose and pH 6.0, without any
supplemental TRP) as standing culture for 3 weeks, this
TRP-degrading bacterium produced IAA in the culture
fluid as a major metabolite at 98 mg l
1 of the cultured
medium (Fig. 4). Since yeast extract generally contains
about 0.3% w/w of TRP, 0.005% yeast extract-amended
medium approximately provides 150 mg l
1 of TRP. If
all the TRP in yeast extract were converted to IAA, the
concentration of IAA would be 128 mg l
1 , almost at the
same level as IAA produced by CK67 cultured without
supplemental TRP. This implies that Serratia sp. CK67
converted almost all the TRP in the medium, even
though the concentration of TRP was only a trace. This
ability might lie in the rhizoplane of host plants.
Since it is known that available cations of essential
metals, including Fe, Cu, Mo, and Zn, are absolutely
Fig. 5. Positive Response of Burkholderia sp. CK43 to CAS-Plate
Assay.
An agar plate (9 cm) containing the CAS-Fe complex showed a
clear blue color, and as a typical positive response on the CAS-plate
assay, CK43 with a yellowish halo-zone around the colony (arrow)
is shown. According to the evaluation shown in the footnote to
Table 3, Burkholderia sp. CK43 is (þþ). The other CK61 and CK67
were negative (
), while CK19 was weak positive (). The circle in
full line on the plate is to emphasize the edges of the bacterial
colonies. The positive responses of CK43 and three more isolates
(Pseudomonas sp. CK2, Burkholderia sp. CK28 and Citrobacter sp.
CK42) were visible within 4 d of inoculation.
poor in fume-rich, woody peat soil,12) the Fe-solubiliz-
ing activities of the rhizobacteria from Fe-chelates are
important in the peatland ecosystem. Among 29 of
bacterial isolates tested by CAS assay for iron-solubi-
lizing activity (Table 3), four isolates, Pseudomonas sp.
CK2, Citrobacter sp. CK42, and two Burkholderia sp.
(CK28 and CK43), formed clear, large yellowish halo-
zones (over 5 mm in diameter). The largest halo-zone
was observed in CK43 as 12 mm diameter around a
3.8-mm bacterial colony, after 10 d of incubation at
25  C (Fig. 5).38) All these active Fe-chelating bacteria
were of the pink type in Salkowski’s reagent test.
Although Payne has reported that most bacterial iron-
chelaters are hydroxamide derivatives,24) the Fe-chelat-
ing agents produced by these CAS assay-positive
bacteria should be characterized.
2208 A. R AHMAN et al.

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